Targeting IRG1 reverses the immunosuppressive function of tumor-associated macrophages and enhances cancer immunotherapy

Immune-responsive gene 1 (IRG1) encodes aconitate decarboxylase (ACOD1) that catalyzes the production of itaconic acids (ITAs). The anti-inflammatory function of IRG1/ITA has been established in multiple pathogen models, but very little is known in cancer. Here, we show that IRG1 is expressed in tumor-associated macrophages (TAMs) in both human and mouse tumors. Mechanistically, tumor cells induce Irg1 expression in macrophages by activating NF-κB pathway, and ITA produced by ACOD1 inhibits TET DNA dioxygenases to dampen the expression of inflammatory genes and the infiltration of CD8+ T cells into tumor sites. Deletion of Irg1 in mice suppresses the growth of multiple tumor types and enhances the efficacy of anti–PD-(L)1 immunotherapy. Our study provides a proof of concept that ACOD1 is a potential target for immune-oncology drugs and IRG1-deficient macrophages represent a potent cell therapy strategy for cancer treatment even in pancreatic tumors that are resistant to T cell–based immunotherapy.


Fig. S1. Positive correlation between IRG1 mRNA expression and TAM fractions in multiple types of human tumors
EPIC (Estimating the Proportions of Immune and Cancer cells) was used to calculated TAM fraction in the indicated types of tumors from the TCGA dataset. Spearman rank correlation test was used to determine the significance of correlation between TAM fraction and IRG1 expression. (B) According to published scRNA-seq data (GEO: GSE154763) in 12 types of human cancer as indicated, the transcripts were re-analyzed. Expression of IRG1 in annotated monocytes and macrophages is illustrated in the UMAP plots. Wild-type BMDMs were co-cultured with different types of syngeneic tumor cells for values were calculated by unpaired, two-tailed Student's t test.
(B) Irg1 induction and ITA accumulation in BMDMs stimulated with TCM. Wild-type BMDMs were treated with conditioned medium from different types of syngeneic tumor cells for 6 or 12 hours. The mRNA expression of Irg1 and intracellular levels of ITA were determined by qRT-PCR and LC-MS, respectively. Data shown are mean ± s.d.
of four independent experiments. The p values were calculated by unpaired, twotailed Student's t test.
(C) B16-F10-TCM with indicated inhibitors were used to stimulate BMDMs for 6 hours.
The mRNA expression of Il-1β, Il-6 and Irg1 were determined by qRT-PCR. Data shown are mean ± s.d. of four independent experiments. The p values were calculated by one-way ANOVA.
(D) RelA occupancy at the promoter region of Irg1 was determined by ChIP-qPCR.
(B and C) BMDMs were challenged with B16-F10-TCM for 12 hours, followed by qRT-PCR to detect the mRNA expression (B) and protein expression (C) of indicated cytokines.
(D and E) BMDMs were co-cultured with B16-F10 (E) or stimulated with B16-F10-TCM (D) for 12 hours, followed by flow cytometry to determine the markers of M1/M2 polarization as indicated.
(F) BMDMs were challenged with B16-F10-TCM for 12 hours, followed by qRT-PCR to detect the mRNA expression of genes which were highly expressed in Vegfa + macrophages.
Data shown are the mean ± s.d. of four (B, D, E, and F) or eight (C) independent experiments. The p values were calculated by two-way ANOVA (B, D and E) and unpaired, two-tailed Student's t test (C). **p < 0.01, ****p < 0.0001 and n.s. denotes not significant. (C) 5hmC enrichment at the promoter regions of indicated genes were determined by hMeDIP-qPCR using BMDMs from Tet2 +/+ and Tet2 HxD mice, as determined by qRT-PCR. The cells were pre-treated with 0.5 mM ITA for 12 hours, followed by treatment with B16-F10-TCM for another 12 hours.
(D) THP1 cells were differentiated to macrophages by treatment with Phorbol 12myristate 13-acetate (PMA, 100 ng/mL) for 24 hours, and then were stimulated with MDA-MB-231-TCM for indicated time, following western-blot to detect IRG1 protein.
(E) IRG1 was depleted in THP-1 cells by using sgRNA and was verified by western blot.
(F) 5hmC enrichment at the promoter regions of indicated genes were determined by hMeDIP-qPCR using THP-1 derived macrophages after treatment with or without MDA-MB-231-TCM for indicated time points, as determined by qRT-PCR. Data are the mean ± s.d. of four or three independent experiments. The p values were calculated by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and n.s.
denotes not significant.

Flow cytometry analysis compared the cytotoxicity of T cells in B16-F10 tumors from
Irg1 +/+ and Irg1 -/mice (n=8 per group). Percentages of cytotoxic CD8 T cells (Ifnγ + Cd8 + ), T cell cytotoxicity (Ifnγ + or Tnfα + of Cd8 + ), exhausted T cells (Pd1 + of Cd8 + ) were measured as described in method. The p values were calculated by an unpaired, two-tailed Student's t test. **p < 0.01, and n.s. denotes not significant.  (B) The percentages of M1-like macrophages (Cd11b + F4/80 + iNos + ) and CD8 + T cells (Cd3 + Cd8 + ) in tumors as described in A were measured. The p values were calculated by one-way ANOVA (A) and unpaired, two-tailed Student's t test (B). *p < 0.05, **p < 0.01, and n.s. denotes not significant. (C) IF staining of indicated markers in pancreatic tumors receiving macrophage therapy as described in Fig. 7C. Scale bar represents 20 μm. The quantification of average cell number was from 5 random high-power fields (HPFs). The p values were calculated by one-way ANOVA. (B and C) IF staining of indicated markers in KPC tumors receiving macrophage therapy. Scale bar represents 50 μm. The quantification of average cell number from 5 random high-power fields (HPFs) is shown. *p < 0.05, **p < 0.01, ***p < 0.001 and n.s. denotes not significant.

Fig. S16. Irg1 acts as a master controller of macrophage antitumor activity
Tumor cells induce Irg1 expression in macrophages through NF-κB activation and subsequently lead to the accumulation of the immunomodulatory metabolite ITA, which dampens the inflammatory response in TAM subsets. As a result, Irg1/ITA alter the functional diversity of TAMs, such as restricted M1-like polarization and reduced pro-angiogenic potential in Vegfa + macrophages, and thereby creates and/or maintains the immunosuppressive TME favorable for tumor growth.